Systems-based approaches enable identification of gene targets which improve the flavour profile of low-ethanol wine yeast strains.

Metabolic engineering has been vital to the development of industrial microbes such as the yeast Saccharomyces cerevisiae. However, sequential rounds of modification are often needed to achieve particular industrial design targets. Systems biology approaches can aid in identifying genetic targets for modification through providing an integrated view of cellular physiology. Recently, research into the generation of commercial yeasts that can produce reduced-ethanol wines has resulted in metabolically-engineered strains of S. cerevisiae that are less efficient at producing ethanol from sugar. However, these modifications led to the concomitant production of off-flavour by-products. A combination of transcriptomics, proteomics and metabolomics was therefore used to investigate the physiological changes occurring in an engineered low-ethanol yeast strain during alcoholic fermentation. Integration of 'omics data identified several metabolic reactions, including those related to the pyruvate node and redox homeostasis, as being significantly affected by the low-ethanol engineering methodology, and highlighted acetaldehyde and 2,4,5-trimethyl-1,3-dioxolane as the main off-flavour compounds. Gene remediation strategies were then successfully applied to decrease the formation of these by-products, while maintaining the 'low-alcohol' phenotype. The data generated from this comprehensive systems-based study will inform wine yeast strain development programmes, which, in turn, could potentially play an important role in assisting winemakers in their endeavour to produce low-alcohol wines with desirable flavour profiles.

Impact of Australian Dekkera bruxellensis strains grown under oxygen-limited conditions on model wine composition and aroma.

Spoilage of red wine by the yeast species Dekkera bruxellensis is a common problem for the global wine industry. When conditions are conducive for growth of these yeasts in wine, they efficiently convert non-volatile hydroxycinnamic acids into aroma-active ethylphenols, thereby reducing the quality of the wine. It has been demonstrated previously that dissolved oxygen is a key factor which stimulates D. bruxellensis growth in wine. We demonstrate that whereas the presence of oxygen accelerates the growth of this species, oxygen-limited conditions favour 4-ethylphenol production. Consequently, we evaluated wine spoilage potential of three D. bruxellensis strains (AWRI1499, AWRI1608 and AWRI1613) under oxygen-limited conditions. Each strain was cultured in a chemically-defined wine medium and the fermentation products were analysed using HPLC and HS-SPME-GC/MS. The strains displayed different growth characteristics but were equally capable of producing ethylphenols. On the other hand, significant differences were observed for 18 of the remaining 33 metabolites analysed and duo-trio sensory analysis indicated significant aroma differences between wines inoculated with AWRI1499 and AWRI1613. When these wines were spiked with low concentrations of 4-ethylphenol and 4-ethylguaiacol, no sensorial differences could be perceived. Together these data suggest that the three predominant D. bruxellensis strains previously isolated during a large survey of Australian wineries do not differ substantively in their capacity to grow in, and spoil, a model wine medium.

Impacts of variations in elemental nutrient concentration of Chardonnay musts on Saccharomyces cerevisiae fermentation kinetics and wine composition.

Chardonnay, being the predominant white wine-grape cultivar in the Australian wine sector, is subject to widely varying winemaking processes with the aim of producing a variety of wine styles. Therefore, juice composition might not always be ideal for optimal fermentation outcomes. Our aim was to better understand the composition of Chardonnay juice and how compositional parameters impact on fermentation outcomes. This was achieved through a survey of 96 commercially prepared Chardonnay juices during the 2009 vintage. Common juice variables were estimated using near infrared spectroscopy, and elemental composition was determined using radial view inductively coupled plasma optical emission spectrometry. The influence of elemental composition on fermentation outcomes was assessed by fermentation of a defined medium formulated to reflect the composition and range of concentrations as determined by the juice survey. Yeast (Saccharomyces cerevisiae) strain effects were also assessed. Key parameters influencing fermentation outcomes were verified by laboratory scale fermentation of Chardonnay juice. This exploration of Chardonnay juice identified interactions between juice pH and potassium concentration as key factors impacting on fermentation performance and wine quality. Outcomes differed depending on yeast strain.

Occurrence of hydrogen sulfide in wine and in fermentation: influence of yeast strain and supplementation of yeast available nitrogen.

Hydrogen sulfide (H2S) is a powerful aroma compound largely produced by yeast during fermentation. Its occurrence in wines and other fermented beverages has been associated with off-odors described as rotten egg and/or sewage. While the formation of hydrogen sulfide (H2S) during fermentation has been extensively studied, it is the final H2S content of wine that is actually linked to potential off-odors. Nevertheless, factors determining final H2S content of wine have received little attention, and it is commonly assumed that high H2S-forming fermentations will result in high final concentrations of H2S. However, a clear relationship has never been established. In this report, we investigated the contribution of yeast strain and nitrogen addition to H2S formation during fermentation and its consequent occurrence the resulting wines. Five commercial Saccharomyces cerevisiae wine yeast strains were used to ferment a Chardonnay juice containing 110 mg/l of YAN (yeast assimilable nitrogen), supplemented with di-ammonium phosphate (DAP) to increase YAN concentration to moderate (260 mg/l) and high (410 mg/l) levels. In contrast to the widely reported decrease in H2S production in response to DAP addition, a non-linear relationship was found such that moderate DAP supplementation resulted in a remarkable increase in H2S formation by each of the five wine yeasts. H2S content of the finished wine was affected by yeast strain, YAN, and fermentation vigor. However, we did not observe a correlation between concentration of H2S in the finished wines and H2S produced during fermentation, with low-forming fermentations often having relatively high final H2S and vice versa. Management of H2S in wine through nitrogen supplementation requires knowledge of initial YAN and yeast H2S characteristics.

Comparison of inorganic and organic nitrogen supplementation of grape juice - Effect on volatile composition and aroma profile of a Chardonnay wine fermented with Saccharomyces cerevisiae yeast.

Inorganic nitrogen salts, and to a growing extent organic nitrogen preparations, are widely used to ameliorate a nitrogen deficiency in wine fermentation, but the impact of nitrogen supplementation on perceived wine sensory profile is essentially unknown. Supplementation of a low nitrogen Chardonnay grape juice with either ammonium nitrogen or combined amino acid and ammonium nitrogen showed that the type of nitrogen and concentration in the range 160-480mgN/l had a substantial impact on the formation of yeast volatile compounds and perceived wine aroma. Addition of amino acid and ammonium nitrogen increased both acetate and medium chain fatty acid esters to a greater extent and decreased higher alcohols to a lesser extent than ammonium nitrogen alone whereas ammonium nitrogen substantially increased ethyl acetate and acetic acid. Low nitrogen wines were rated relatively low in floral/fruity aroma descriptors, while moderate nitrogen wines showed a good balance between desirable and less desirable attributes, whereas high nitrogen produced either an acetic/solvent character or highest ratings for floral/fruity attributes, depending on nitrogen type. These results show that amount and type of nitrogen supplement can substantially modulate Chardonnay wine volatiles composition and perceived aroma.

Hanseniaspora vineae and the Concept of Friendly Yeasts to Increase Autochthonous Wine Flavor Diversity.

In this perspective, we will explain the concept of "friendly" yeasts for developing wine starters that do not suppress desirable native microbial flora at the initial steps of fermentation, as what usually happens with Saccharomyces strains. Some non-Saccharomyces strains might allow the development of yeast consortia with the native terroir microflora of grapes and its region. The positive contribution of non-Saccharomyces yeasts was underestimated for decades. Avoiding them as spoilage strains and off-flavor producers was the main objective in winemaking. It is understandable, as in our experience after more than 30 years of wine yeast selection, it was shown that no more than 10% of the isolated native strains were positive contributors of superior flavors. Some species that systematically gave desirable flavors during these screening processes were Hanseniaspora vineae and Metschnikowia fructicola. In contrast to the latter, H. vineae is an active fermentative species, and this fact helped to build an improved juice ecosystem, avoiding contaminations of aerobic bacteria and yeasts. Furthermore, this species has a complementary secondary metabolism with S. cerevisiae, increasing flavor complexity with benzenoid and phenylpropanoid synthetic pathways practically inexistent in conventional yeast starters. How does H. vineae share the fermentation niche with other yeast strains? It might be due to the friendly conditions it creates, such as ideal low temperatures and low nitrogen demand during fermentation, reduced synthesis of medium-chain fatty acids, and a rich acetylation capacity of aromatic higher alcohols, well-known inhibitors of many yeasts. We will discuss here how inoculation of H. vineae strains can give the winemaker an opportunity to develop ideal conditions for flavor expression of the microbial terroir without the risk of undesirable strains that can result from spontaneous yeast fermentations.

Acetic acid bacteria spoilage of bottled red wine -- a review.

Acetic acid bacteria (AAB) are ubiquitous organisms that are well adapted to sugar and ethanol rich environments. This family of Gram-positive bacteria are well known for their ability to produce acetic acid, the main constituent in vinegar. The oxidation of ethanol through acetaldehyde to acetic acid is well understood and characterised. AAB form part of the complex natural microbial flora of grapes and wine, however their presence is less desirable than the lactic acid bacteria and yeast. Even though AAB were described by Pasteur in the 1850s, wine associated AAB are still difficult to cultivate on artificial laboratory media and until more recently, their taxonomy has not been well characterised. Wine is at most risk of spoilage during production and the presence of these strictly aerobic bacteria in grape must and during wine maturation can be controlled by eliminating, or at least limiting oxygen, an essential growth factor. However, a new risk, spoilage of wine by AAB after packaging, has only recently been reported. As wine is not always sterile filtered prior to bottling, especially red wine, it often has a small resident bacterial population (<10(3) cfu/mL), which under conducive conditions might proliferate. Bottled red wines, sealed with natural cork closures, and stored in a vertical upright position may develop spoilage by acetic acid bacteria. This spoilage is evident as a distinct deposit of bacterial biofilm in the neck of the bottle at the interface of the wine and the headspace of air, and is accompanied with vinegar, sherry, bruised apple, nutty, and solvent like off-aromas, depending on the degree of spoilage. This review focuses on the wine associated AAB species, the aroma and flavour changes in wine due to AAB metabolism, discusses the importance of oxygen ingress into the bottle and presents a hypothesis for the mechanism of spoilage of bottled red wine.

The role of lysine amino nitrogen in the biosynthesis of mousy off-flavor compounds by Dekkera anomala.

Mousy off-flavor is an insidious and economically disastrous microbiologically derived spoilage characteristic of wine and other fermented beverages. Tainted wines are rendered unpalatable and there is currently no satisfactory procedure for removal of the off-flavor. Here we report the confirmation of that both d- and l-lysine can act as a precursor for the formation of mousy off-flavor N-heterocycles. Further, through the use of stable isotope feeding experiments, we could establish that a pentylamine group from lysine is incorporated into the piperideine moiety of two off-flavor N-heterocycles. A biochemical pathway for the formation of mousy off-flavor compounds is proposed.

Hydrolysis and transformation of grape glycosidically bound volatile compounds during fermentation with three Saccharomyces yeast strains.

The ability of three Saccharomyces wine yeasts (S. cerevisiae AWRI 838, S. cerevisiae AWRI 1537, and S. bayanus AWRI 1375) to liberate volatile compounds from sugar-bound aroma precursors was investigated using synthetic and grape glycosides under different experimental conditions. In model systems involving the incubation of yeast cells with either synthetic or grape-derived glycosides under conditions more favorable for glycosidase activities and less favorable for acid-catalyzed hydrolysis (pH 5.0 and 30 degrees C), all yeast strains studied proved to be capable of hydrolyzing glycosides, with S. bayanus AWRI 1375 displaying greater hydrolytic activity than S. cerevisiae AWRI 838 and AWRI 1537. During the fermentation of a chemically defined grape juice-like medium containing glycosidic precursors extracted from Vitis vinifera cv. White Frontignac (synonym Muscat à Petit Grains Blanc), all yeasts promoted a significant hydrolysis of different precursors, which varied according to the chemical structures of both the sugar and the aglycon moieties, as determined by GC-MS analysis of trifluoroacetylated derivatives. Hydrolysis of the White Frontignac derived glycosidic precursors during fermentation resulted in the release of monoterepene alcohols, terpene oxides, terpene diols, and 3-oxo-alpha-ionol, demonstrating the significant potential of these yeast strains to contribute to wine varietal volatile composition during alcoholic fermentation.

De novo synthesis of monoterpenes by Saccharomyces cerevisiae wine yeasts.

This paper reports the production of monoterpenes, which elicit a floral aroma in wine, by strains of the yeast Saccharomyces cerevisiae. Terpenes, which are typical components of the essential oils of flowers and fruits, are also present as free and glycosylated conjugates amongst the secondary metabolites of certain wine grape varieties of Vitis vinifera. Hence, when these compounds are present in wine they are considered to originate from grape and not fermentation. However, the biosynthesis of monoterpenes by S. cerevisiae in the absence of grape derived precursors is shown here to be of de novo origin in wine yeast strains. Higher concentration of assimilable nitrogen increased accumulation of linalool and citronellol. Microaerobic compared with anaerobic conditions favored terpene accumulation in the ferment. The amount of linalool produced by some strains of S. cerevisiae could be of sensory importance in wine production. These unexpected results are discussed in relation to the known sterol biosynthetic pathway and to an alternative pathway for terpene biosynthesis not previously described in yeast.

The 'buttery' attribute of wine--diacetyl--desirability, spoilage and beyond.

The diketone, diacetyl, is a major flavour metabolite produced by lactic acid bacteria (LAB). Of the LAB associated with wine, Oenococcus oeni is encouraged during the malolactic (ML) fermentation, a biodeacidification of wine during which the metabolism of diacetyl occurs. Diacetyl, which imparts a buttery aroma and flavour to many fermented foods and beverages, is a key flavour compound of most fermented dairy products. In wine, diacetyl has important stylistic implications. The biosynthesis of diacetyl is dependent upon citric acid metabolism and diacetyl is an intermediate metabolite which can be further reduced to acetoin and the alcohol, 2,3-butanediol. This review will focus on the sensory perception, metabolism, genetics and analysis of diacetyl during wine production. The extensive knowledge of diacetyl metabolism in dairy LAB is used to enhance the understanding of diacetyl metabolism of wine LAB. Factors which can effect the formation and concentration of diacetyl in wine are discussed. These include malolactic bacterial strain, wine chemical and physical parameters (pH, temperature, citric acid, sulfur dioxide, aeration) and the presence of yeast lees. Finally, the affects of other wine components, such as phenolics, are discussed.

Mousy off-flavor of wine: precursors and biosynthesis of the causative N-heterocycles 2-ethyltetrahydropyridine, 2-acetyltetrahydropyridine, and 2-acetyl-1-pyrroline by Lactobacillus hilgardii DSM 20176.

The N-heterocyclic bases, 2-ethyltetrahydropyridine (1), 2-acetyl-1-pyrroline (2), and 2-acetyltetrahydropyridine (3) are associated with the occurrence of mousy off-flavor in wine. The biosynthesis of these N-heterocycles by the wine lactic acid bacterium, Lactobacillus hilgardii DSM 20176, was studied by high-cell-density incubation in combination with a minimal chemically defined N-heterocycle assay medium. The key components of the defined N-heterocycle assay medium included D-fructose, ethanol, L-lysine, L-ornithine, and mineral salts. N-heterocycle formation was quantitatively determined by gas chromatography-mass spectrometry. The formation of 2 and 3 required the concomitant availability of a fermentable carbohydrate (D-fructose), ethanol, and iron (Fe(2+)). In addition, L-ornithine stimulated the formation of 2 and repressed 3 formation, whereas L-lysine stimulated the formation of 3 and repressed 2 formation. Incorporation of d(6)-ethanol into the acetyl side chain of 2 and 3, and of d(4)-acetaldehyde into the acetyl side chain of 3, confirmed that ethanol and acetaldehyde could serve as major side chain precursors. A pathway for the formation of 2 and 3 by heterofermentative lactic acid bacteria is proposed involving the interaction of accumulated C-2 intermediates from the heterolactic pathway and N-heterocyclic intermediates derived from the metabolism of L-ornithine and L-lysine.

Effects of rehydration nutrients on H2S metabolism and formation of volatile sulfur compounds by the wine yeast VL3.

In winemaking, nutrient supplementation is a common practice for optimising fermentation and producing quality wine. Nutritionally suboptimal grape juices are often enriched with nutrients in order to manipulate the production of yeast aroma compounds. Nutrients are also added to active dry yeast (ADY) rehydration media to enhance subsequent fermentation performance. In this study we demonstrate that nutrient supplementation at rehydration also has a significant effect on the formation of volatile sulfur compounds during wine fermentations. The concentration of the 'fruity' aroma compounds, the polyfunctional thiols 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA), was increased while the concentration of the 'rotten egg' aroma compound, hydrogen sulfide (H2S), was decreased. Nutrient supplementation of the rehydration media also changed the kinetics of H2S production during fermentation by advancing onset of H2S production. Microarray analysis revealed that this was not due to expression changes within the sulfate assimilation pathway, which is known to be a major contributor to H2S production. To gain insight into possible mechanisms responsible for this effect, a component of the rehydration nutrient mix, the tri-peptide glutathione (GSH) was added at rehydration and studied for its subsequent effects on H2S formation. GSH was found to be taken up during rehydration and to act as a source for H2S during the following fermentation. These findings represent a potential approach for managing sulfur aroma production through the use of rehydration nutrients.

Comparison of three methods for accurate quantification of hydrogen sulfide during fermentation.

Two analytical approaches for the rapid measurement of hydrogen sulfide (H(2)S) have been compared to a reference method for their potential application as a rapid procedure for the quantification of H(2)S formed during alcoholic fermentations. In one case, silver nitrate, lead acetate, and mercuric chloride selective detector tubes for the analysis of H(2)S in air were investigated. In the other case, a commercially available kit for the diagnosis of nitrogen starvation in wine fermentations, which is based on the detection of H(2)S, was investigated. Both methods exhibited excellent linearity of response, but the mercuric chloride tube was found to suffer from interferences due to the concomitant presence of mercaptans, which resulted in erroneous H(2)S quantification. A comparative study between the two methods studied and the cadmium hydroxide/methylene blue reference method commonly used to monitor H(2)S indicate that the two new methods displayed better recoveries at low H(2)S concentrations, besides being more rapid and economical. The two new methods were successfully used to quantify production of H(2)S in different grape juice fermentations. The suitability of each method for the study of specific aspects of H(2)S production during fermentation is discussed.

Volatile composition and sensory properties of Shiraz wines as affected by nitrogen supplementation and yeast species: rationalizing nitrogen modulation of wine aroma.

The effects of yeast assimilable nitrogen (YAN) supplementation on Shiraz volatile composition and sensory properties have been investigated. A low YAN Shiraz must (YAN 100 mg/L) was supplemented with nitrogen in the form of diammonium phosphate (DAP) to a final YAN of either 250 or 400 mg/L. Fermentation was carried out with either Saccharomyces cerevisiae or Saccharomyces bayanus , with maceration on skins. For both yeast strains, high DAP additions increased the ratings of positive sensory attributes such as "red fruit" and "dark fruit" and decreased the "yeast/cheese", "vegetal", and "earth/dirty" attributes. For the S. cerevisiae yeast moderate DAP addition resulted in higher "reduced" attribute scores. DAP supplementation had a strong influence on formation of acetates, fatty acid ethyl esters, higher alcohols, hydrogen sulfide, ethyl mercaptan, methyl mercaptan, DMS, and DES. Partial least-squares regression analysis of chemical and sensory data indicated that esters, sulfides, and mercaptans were associated with fruit-related descriptors, whereas hydrogen sulfide was associated with the "reduced" attribute. Nitrogen-related variations in the concentration of other yeast metabolites such as ethanol and 2- and 3-methylbutanoic acids also affected perceived fruitiness. Depending on yeast species DAP supplementation to a low nitrogen must can result in increased reduction off-odor.

Effect of nitrogen supplementation and Saccharomyces species on hydrogen sulfide and other volatile sulfur compounds in shiraz fermentation and wine.

A Shiraz must with low yeast assimilable nitrogen (YAN) was supplemented with two increasing concentrations of diammonium phosphate (DAP) and fermented with one Saccharomyces cerevisiae and one Saccharomyces bayanus strain, with maceration on grape skins. Hydrogen sulfide (H(2)S) was monitored throughout fermentation, and a total of 16 volatile sulfur compounds (VSCs) were quantified in the finished wines. For the S. cerevisiae yeast strain, addition of DAP to a final YAN of 250 or 400 mg/L resulted in an increased formation of H(2)S compared to nonsupplemented fermentations (100 mg/L YAN). For this yeast, DAP-supplemented fermentations also showed prolonged formation of H(2)S into the later stage of fermentation, which was associated with increased H(2)S in the final wines. The S. bayanus strain showed a different H(2)S production profile, in which production was inversely correlated to initial YAN. No correlation was found between total H(2)S produced by either yeast during fermentation and H(2)S concentration in the final wines. For both yeasts, DAP supplementation yielded higher concentrations of organic VSCs in the finished wines, including sulfides, disulfides, mercaptans, and mercaptoesters. PCA analysis indicated that nitrogen supplementation before fermentation determined a much clearer distinction between the VSC profiles of the two yeasts compared to nonsupplemented fermentations. These results raise questions concerning the widespread use of DAP in the management of low YAN fermentations with respect to the formation of reductive characters in wine.

Volatile and color composition of young and model-aged Shiraz wines as affected by diammonium phosphate supplementation before alcoholic fermentation.

A Shiraz must with low yeast assimilable nitrogen (YAN) was supplemented with two concentrations of diammonium phosphate (DAP) and then fermented with maceration on grape skins. The nonvolatile, volatile, and color composition of the final wines were investigated. Ethanol and residual sugars were not affected by DAP supplementation, while glycerol, SO 2, and residual YAN increased and acetic acid decreased. DAP-supplemented treatments gave rise to higher concentrations of acetates, fatty acids, and fatty acid ethyl esters but lower concentrations of branched-chain fatty acids and their ethyl esters. No major difference between treatments was observed for higher alcohols, monoterpenes, norisoprenoids, and low-molecular-weight sulfur compounds. DAP-supplemented fermentations resulted in wines with higher concentrations of malvidin-3-glucoside, higher color intensity, and altered color tonality. Model aging studies indicated that higher concentrations of esters are still present in wines from the DAP-treated fermentations after aging. DAP supplementation also resulted in increased concentrations of dimethyl sulfide after model aging. It can be concluded that DAP treatment of a low YAN must fermented by maceration on skins can significantly affect wine color, aroma, and flavor.

Production of fermentation aroma compounds by Saccharomyces cerevisiae wine yeasts: effects of yeast assimilable nitrogen on two model strains.

The contribution of yeast fermentation metabolites to the aromatic profile of wine is well documented; however, the biotechnological application of this knowledge, apart from strain selection, is still rather limited and often contradictory. Understanding and modeling the relationship between nutrient availability and the production of desirable aroma compounds by different strains must be one of the main objectives in the selection of industrial yeasts for the beverage and food industry. In order to overcome the variability in the composition of grape juices, we have used a chemically defined model medium for studying yeast physiological behavior and metabolite production in response to nitrogen supplementation so as to identify an appropriate yeast assimilable nitrogen level for strain differentiation. At low initial nitrogen concentrations, strain KU1 produced higher quantities of esters and fatty acids whereas M522 produced higher concentrations of isoacids, gamma-butyrolactone, higher alcohols and 3-methylthio-1-propanol. We propose that although strains KU1 and M522 have a similar nitrogen consumption profile, they represent useful models for the chemical characterization of wine strains in relation to wine quality. The differential production of aroma compounds by the two strains is discussed in relation to their capacity for nitrogen usage and their impact on winemaking. The results obtained here will help to develop targeted metabolic footprinting methods for the discrimination of industrial yeasts.

Genetic diversity of Dekkera bruxellensis yeasts isolated from Australian wineries.

Yeasts of the genus Dekkera and its anamorph Brettanomyces represent a significant spoilage issue for the global wine industry. Despite this, there is limited knowledge of genetic diversity and strain distribution within wine and winery-related environments. In this study, amplified fragment length polymorphism (AFLP) analysis was conducted on 244 Dekkera bruxellensis isolates from red wine made in 31 winemaking regions of Australia. The results indicated there were eight genotypes among the isolates, and three of these were commonly found across multiple winemaking regions. Analysis of 26S rRNA gene sequences provided further evidence of three common, conserved groups, whereas a phylogeny based upon the AFLP data demonstrated that the most common D. bruxellensis genotype (I) in Australian red wine was highly divergent from the D. bruxellensis type strain (CBS 74).

Metabolic profiling as a tool for revealing Saccharomyces interactions during wine fermentation.

The multi-yeast strain composition of wine fermentations has been well established. However, the effect of multiple strains of Saccharomyces spp. on wine flavour is unknown. Here, we demonstrate that multiple strains of Saccharomyces grown together in grape juice can affect the profile of aroma compounds that accumulate during fermentation. A metabolic footprint of each yeast in monoculture, mixed cultures or blended wines was derived by gas chromatography - mass spectrometry measurement of volatiles accumulated during fermentation. The resultant ion spectrograms were transformed and compared by principal-component analysis. The principal-component analysis showed that the profiles of compounds present in wines made by mixed-culture fermentation were different from those where yeasts were grown in monoculture fermentation, and these differences could not be produced by blending wines. Blending of monoculture wines to mimic the population composition of mixed-culture wines showed that yeast metabolic interactions could account for these differences. Additionally, the yeast strain contribution of volatiles to a mixed fermentation cannot be predicted by the population of that yeast. This study provides a novel way to measure the population status of wine fermentations by metabolic footprinting.